A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany.
PLoS Biology (Impact Factor: 9.34). 06/2010; 8(6):e1000408. DOI: 10.1371/journal.pbio.1000408
Source: PubMed


DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

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    • "However, its functional importance was highlighted in a recent study of AP-5 mutations in patients. Notably, mutation in ζ subunit also causes HSP disease [77], indicating AP-4 and AP-5 may sort the same cargo proteins or both important for axonal maintenance [78]. "
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    ABSTRACT: The Adaptor Protein (AP) complexes are heterotetrameric protein complexes that mediate intracellular membrane trafficking along endocytic and secretory transport pathways. There are five different AP complexes: AP-1, AP-2 and AP-3 are clathrin-associated complexes, while AP-4 and AP-5 are not. These five AP complexes localize to different intracellular compartments and mediate membrane trafficking in distinct pathways. They recognize and concentrate cargo proteins into vesicular carriers that mediate transport from a donor membrane to a target organellar membrane. AP complexes play import roles in maintaining the normal physiological function of eukaryotic cells. Dysfunction of AP complexes has been implicated in a variety of inherited disorders, including: MEDNIK syndrome, Fried syndrome, Hermansky-Pudlak syndrome (HPS) and hereditary spastic paraplegia (HSP).
    Bioscience Reports 06/2014; 34(4). DOI:10.1042/BSR20140069 · 2.64 Impact Factor
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    • "Disruption of spg11 (SPG11, spatacsin) or zfyve26 (SPG15, spastizin) expression during zebrafish development induced a range of developmental defects, including locomotor impairment and abnormal architecture of spinal MN axons (Martin et al., 2012; Southgate et al., 2010). These two proteins are components of a multi-protein complex (Słabicki et al., 2010) and AMOs targeting the two genes at the same time suggested that they were involved in a pathway required for spinal MN axon outgrowth (Martin et al., 2012). However, an EMS (Raldú a et al., 2008) was apparently induced by changing the expression of these genes, thus making it difficult to interpret the phenotype, which may also be due to a developmental delay. "
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    ABSTRACT: Motor neuron diseases (MNDs) are an etiologically heterogeneous group of disorders of neurodegenerative origin, which result in degeneration of lower (LMNs) and/or upper motor neurons (UMNs). Neurodegenerative MNDs include pure hereditary spastic paraplegia (HSP), which involves specific degeneration of UMNs, leading to progressive spasticity of the lower limbs. In contrast, spinal muscular atrophy (SMA) involves the specific degeneration of LMNs, with symmetrical muscle weakness and atrophy. Amyotrophic lateral sclerosis (ALS), the most common adult-onset MND, is characterized by the degeneration of both UMNs and LMNs, leading to progressive muscle weakness, atrophy, and spasticity. A review of the comparative neuroanatomy of the human and zebrafish motor systems showed that, while the zebrafish was a homologous model for LMN disorders, such as SMA, it was only partially relevant in the case of UMN disorders, due to the absence of corticospinal and rubrospinal tracts in its central nervous system. Even considering the limitation of this model to fully reproduce the human UMN disorders, zebrafish offer an excellent alternative vertebrate model for the molecular and genetic dissection of MND mechanisms. Its advantages include the conservation of genome and physiological processes and applicable in vivo tools, including easy imaging, loss or gain of function methods, behavioral tests to examine changes in motor activity, and the ease of simultaneous chemical/drug testing on large numbers of animals. This facilitates the assessment of the environmental origin of MNDs, alone or in combination with genetic traits and putative modifier genes. Positive hits obtained by phenotype-based small-molecule screening using zebrafish may potentially be effective drugs for treatment of human MNDs.
    Progress in Neurobiology 04/2014; 118. DOI:10.1016/j.pneurobio.2014.03.001 · 9.99 Impact Factor
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    • "High-throughput screening approaches have been mostly confined to searching for genes, which modulate HDR efficiency (17,18,19,20). Such screens are generally based on siRNA techniques and often result in a relatively high false positive rate due to non-specific silencing of genes, as was recently demonstrated (18). "
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    ABSTRACT: DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy.
    Nucleic Acids Research 03/2014; 42(9). DOI:10.1093/nar/gku217 · 9.11 Impact Factor
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